• Molecular & Cellular Toxicology
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• v.1 no.2
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• pp.92-98
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• 2005

DNA microarray analysis of gene expression in toxicogenomics typically requires relatively large amounts of total RNA. This limits the use of DNA microarray when the sample available is small. To confront this limitation, different methods of linear RNA amplification that generate antisense RNA (aRNA) have been optimized for microarray use. The target preparation strategy using amplified RNA in DNA microarray protocol can be divided into direct-incorporation labeling which resulted in cDNA targets (Cy-dye labeled cDNA from aRNA) and indirect-labeling which resulted in cRNA targets (i.e. Cy-dye labeled aRNA), respectively. However, despite the common use of amplified targets (cDNA or cRNA) from aRNAs, no systemic assessment for the use of amplified targets and bias in terms of hybridization performance has been reported. In this investigation, we have compared the hybridization performance of cRNA targets with cDNA targets from aRNA on a 10 K cDNA microarrays. Under optimized hybridization conditions, we found that 43% of outliers from cDNA technique and 86% from the outlier genes were reproducibly detected by both targets hybridization onto cDNA microarray. This suggests that the cRNA labeling method may have a reduced capacity for detecting the differential gene expression when compared to the cDNA target preparation. However, further validation of this discordant result should be pursued to determine which techniques possesses better accuracy in identifying truly differential genes.

• Journal of fish pathology
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• v.7 no.2
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• pp.95-103
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• 1994

We have prepared cDNA libray of loach. M. mizolepis in order to isolate cDNA clone of growth hormone gene. Total RNA was isolated from pituitary of loach, and then mRNA was further purified from total RNA by oligo (dT)-coupled magnetic beads. The purified mRNA was used as substrates to prepare cDNA. The resulting cDNA was ligated into the EcoRV/Smal site of pBlueKS+. The ligation mixture have transformed E. coli JM109 strain with electroporator to obtain high yield of transformation efficiency. All the transformants was screened with DIG-labeled Tilapia growth hormone gene by high density colony hybridization. After isolating 10 putative colonies showing the positive signals, secondary colony hybridization and southern hybridization could confirm it as true clones. The nucleotide sequence of one candidate, pCGHI, was compared with 312 bp DNA fragment used as DNA probe and show 52% relative homology to Tilapia growth hormone gene.

• Korean Journal of Food Science and Technology
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• v.35 no.3
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• pp.470-474
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• 2003

DNA sequence information on small-subunit rRNA gene (16S rDNA) obtained from food-poisoning bacterial culture was used to investigate the presence of bacterial pathogens in food. By reverse dot blot detection method, presence of food-poisoning bacteria could be confirmed on hybridization of digoxigenin-labeled 16S rDNA Polymerase Chain Reaction (PCR) primer product and biotin-labeled specific oligonucleotide probe. Escherichia coli, Bacillus cereus. and Salmonella sp. were used as the representative food-poisoning bacterial microorganisms. An oligonucleotide probe, based on the variable region of 16S rRNA gene, was used as the specific probe. These tools may be more useful than classic biochemical method for rapid identification of contaminated food.

• YAKHAK HOEJI
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• v.33 no.5
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• pp.300-307
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• 1989

Expression of transferrin gene in various organs of rat was studied using rat transferrin cDNA. The hybridization method of $[^{35}S]-labeled$ transferrin cDNA with transferrin mRNA in cytoplasmic preparations was used to measure the level of transferrin mRNA. The rat from 15-day old fetus to 21-day old postnatal were employed as an animal model. In the liver, the level of transferrin mRNA increased with increasing age. However, the level of transferrin mRNA in brain was significantly lower than that in liver and the level did not increase with age.

• Tuberculosis and Respiratory Diseases
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• v.39 no.3
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• pp.242-247
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• 1992

Background: The ability to precisely measure specific mRNA levels by hybridization to complementary DNA probes is an important tool for analyzing the regulation of gene expression. Surfactant proteins have important roles in regulating surfactant metabolism as well as in determing its physical properties. Method: The complete coding regions for rat surfactant protein complementary DNA of surfactant protein B were subcloned into pGem 3Z or 4Z such that either antisense or sense transcripts were obtained by using SP 6 RNA polymerase. Surfactant protein B mRNA was measured by filter hybridization. Results: Equation of standard curve between counts per minute (Y) and surfactant protein B mRNA transcript input (X) was Y=2034.9 X+159.1. Correlation coefficient was 1.0. Couclusions: Filter hybridization assay is suited to situation when rapid, accurate quantitation of multiple samples is required.

• Journal of Plant Biology
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• v.38 no.2
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• pp.203-209
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• 1995

Tobacco (Nicotiana tahacum L. cv. Wisconsin 38) leaf discs were cocultivated with Agrohacterium carrying a leghemoglobin (Lb) cDNA from Canavalia lineata. Seven plants were regenerated from the transformed leaf discs on MS media supplemented with 0.5 mg/L BAP, 0.1 mg/L ${\alpha}-NAA$, 200 mg/L kanamycin and 500 mg/L carbenicillin. Southern hybridization and PCR of genomic DNA from transgenic plants showed that the Lb cDNA was stably integrated into the genome of the tobacco. Total RNA from the transgenic tobacco showed northern hybridization signal at 1,000 nt and PCR of the first strand cDNA synthesized from the total RNA amplified 0.5 kb Lb cDNA. Furthermore, western hybridization using a polyclonal antibody against soybean Lb showed a 15.8 kD LB-like band on SDS-PAGE of proteins from the transformed tobacco. These results demonstrated that the Lb cDNA of C. lineata was not only incorporated into the genome of tobacco, but also transcribed into mRNA and translated into Lb protein in the transformed tabacco.

• Korean Journal of Veterinary Research
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• v.32 no.3
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• pp.389-398
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• 1992

Molecular cloning was carried out on the Danish strain of bovine viral diarrhea virus(BVDV) to construct strategy for the diagnostic tools and effective vaccine of BVD afterwards. A recombinant DNA clone(No. 29) was established successfully from cDNA for viral RNA tailed with adenine homopolymer at 3'-end. $^{32}P$-labeled DNA probes of 300~1,800bp fragments, originating from the clone 29, directed specific DNA-RNA hybridization results with BVDV RNA. Recombinant DNA of the clone 29 was about 5,200bp representing 41.6% of the full length of Danish strain's RNA, and restriction sites were recognized for EcoR I, Sst I, Hin d III and Pst I restriction enzymes in the DNA fragment.

• Tuberculosis and Respiratory Diseases
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• v.51 no.6
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• pp.517-529
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• 2001

Background : Surfactant protein C(SP-C) is a hydrophobic 5,000 dalton molecule. SP-C has the primary roles in accelerating surface spreading of a surfactant phospholipid. The filter hybridization and solution hybridization assays are both rapid and sensitive and can be used to measure the RNAs complementary to any cloned DNA sequence. Methods : The authors measured the SP-C mRNA levels quantitatively using solution hybridization and filter hybridization assays to obtain a standard curve equation to quantify the mRNA of unknown samples comparatively. Results : 1. The minimum level of the specimens by solution hybridization was 3 pg for SP-C mRNA. 2. The standard curve equation of the solution hybridization assay between the counts per minute(Y) and the SP-C mRNA transcript input(X) was Y=6.46 X+244. The correlation coefficient was 0.99. 3. The minimum detection level of specimens by filter hybridization was 0.1 ng for SP-C mRNA. 4. The standard curve equation of the filter hybridization assay between the counts per minute(Y) and SP-C mRNA transcript input(X) is Y=2541.6 X+252.7. The correlation coefficient was 0.99. Conclusions : A comparison of CPM/filter in the linear range allowed an accurate and reproducible estimation of the SP-C mRNA copy number. Filter hybridization and solution hybridization assays are both rapid and sensitive and can be used to measure the RNAs complementary to any cloned DNA sequence. It is ideally suited to situations where accurate quantitation of multiple samples is required.

• Korean Journal of Microbiology
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• v.21 no.4
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• pp.229-237
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• 1983

Total tRNA genes from Aspergillus nidulans were cloned for the further investigation of the structure and expression of Aspergillus tRNA genes. Aspergillus DNA was isolated from spores and cloned into pBR322 plasmid DNA using BamHI and $T_4$ ligase. The recombinant hybrid DNA was transformed into E. coli HB101 and some 30,000 transformants were initially selected. Of these, about 5,300 E. coli clones containing Aspergillus DNA inserted into plasmid pBR322 at BamHl site have been isolated. The hybridization data obtained from the labeled Aspergillus $^{32}P-tRNA$ indicated that 105 colonies carried the total tRNA genes. From the data above and cohybridization experiment, tRNA genes of Aspergillus nidulans seem to be twice more clustered than those of yeast.

• KSBB Journal
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• v.22 no.6
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• pp.387-392
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• 2007

Recently, microfluidic biochips for DNA microarray are providing a number of advantages such as, reduction in reagent volume, high-throughput parallel sample screening, automation of processing, and reduction in hybridization time. Particularly, the enhancement of target probe hybridization by decrease of hybridization time is an important aspect highlighting the advantage of microfluidic DNA microarray platform. Fundamental issues to overcome extremely slow diffusion-limited hybridization are based on physical, electrical or fluidic dynamical mixing technology. So far, there have been some reports on the enhancement of the hybridization with the microfluidic platforms. In this review, their principle, performance, and outreaching of the technology are overviewed and discussed for the implementation into many bio-applications.